Analysis of polyhydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA containing 3‐hydroxy‐2‐methylvalerate

Abstract

The identification of phaC which encodes PHA synthase, that is involved in the formation of polyhydroxyalkanoate (PHA) containing 3-hydroxy-2-methylvalerate (3H2MV), was attempted. As of now, PHA containing 3H2MV has been reported to be produced only by mixed microbial cultures in activated sludge, but no pure bacterial cultures. A laboratory-scale activated sludge process was operated for 67 days. During the operation of the activated sludge process, its capacity to produce PHA containing 3H2MV, and the diversity of the partial phaC genes in the activated sludge microorganisms were monitored periodically. Analysis of the partial phaC genes was conducted by PCR followed by cloning and DNA sequencing, or by PCR followed by terminal-restriction fragment length polymorphism (T-RFLP). The cloning-sequencing of the 263 clones gave 11 distinct genetic groups (GGs). All of the genetic groups had similarities to known phaC higher than 48%, and one of them had similarity as high as 96% to that of Alcaligenes sp. The behavior of each of the genetic groups during the operation of the activated sludge process was monitored by the T-RFLP method. The restriction enzyme AccII, with the help of MboI, enabled the monitoring of each of the genetic groups. One of the genetic groups was found to have a strong correlation with the capability of the activated sludge to produce PHA containing 3H2MV, and its DNA sequence together with its amino acid sequence are reported.

Unveiling PHA-storing populations using molecular methods

“…After day 39, A[152] disappeared alongside with 3H2MV production, strongly suggesting that the bacterial group with A[152] was responsible for the production of PHA with a high content of 3H2MV with the closest protein suggested by Basic Local Alignment Search Tool (BLAST) analysis belonging to Pseudomonas sp. class II PHA synthase (Michinaka et al 2007).…”

“…The T-RFLP technique was used by Michinaka et al (2007) to investigate the bacteria able to accumulate PHA containing 3H2MV in a MMC selected in an AN/AE SBR operated for 67 days, with 10 days of SRT, and fed with synthetic wastewater containing propionate/acetate (14:3) as carbon sources. The SBR showed an increase in the amount of 3H2MV from 6 to 38 %.…”

Biosynthesis and characterization of novel poly(3-hydroxybutyrate-co-3-hydroxy-2-methylbutyrate): thermal behavior associated with α-carbon methylation

“…The composition of other samples were determined in the same manner, and summarized in Table 2. Additional structural evidence for incorporation of 3H2MB was obtained in 13 C-NMR spectra at 125 MHz. In Fig.…”

“…13 In this study, biosynthesis of 3H2MB-containing copolymers from tiglic acid was achieved by expressing the enzyme PhaC Ac (from A. caviae) in E. coli LS5218, which is a mutant strain that utilizes fatty acids more efficiently than other E. coli strains.…”

“…1 H-NMR data were collected at 23 C with a 7.2 ms pulse width, 90 pulse angle, 5 s pulse repetition, 6000 Hz spectra width, and 16K data points. 13 C-NMR data were collected at 25 C with a 6.1 ms pulse width, 90 pulse angle, 5 s pulse repetition, 27 000 Hz spectra width, and 33K data points. Tetramethylsilane (Me 4 Si) was used as internal chemical shi standard.…”